Method for the production of a heterologous protein by a fungus

ABSTRACT

Production of heterologous protein by fungal cells is good when the feeding medium comprises a carbon source comprising 50 to 100 wt % of ethanol and an inducer.

FIELD OF THE INVENTION

The invention relates to a method for the production of a heterologousprotein or peptide in a fungus.

BACKGROUND OF THE INVENTION

Heterologous protein production in a host such as a fungus is wellknown. EP-A-481,008 discloses production of a heterologous protein in ayeast, which is grown on glucose.

The industrial, large scale production of a heterologous protein by ahost organism in a fed batch fermentation generally shows three phases.

The first phase is the batch phase which is defined as the phase whereinthe cells are grown to the required concentration. In this phase thecells are grown exponentially. Models describing the batch phase assumethat cells do not die, that oxygen is present in excess, and that allother conditions are such that growth can occur unlimited. This impliesthat in the batch phase all nutrient requirements are supplied insufficient quantity. In summary the batch phase is the phase where cellsare amplified while (heterologous) protein production is still low.

The second phase is the feed phase which is defined as the phase whereincarbon source and other requirements are fed to a fermenter in arelatively concentrated liquid stream at a precalculated rate, the “feedrate”. In this phase emphasis is on protein production by the growncells and cell growth leading to an increase of biomass. Substrate thatis fed to the fermenter is at this stage used generally for cell growthand product synthesis. The cell growth is controlled by the feed rate toobtain an optimum in cell growth and production of eterologous protein.

Eventually the third phase is reached which is defined as the declinephase, wherein limiting conditions arise. In this phase for exampleoxygen concentration in the fermenter is low to zero, leading in somecases to formation of ethanol. In this stage, most cells will focus onmaintenance and usually product synthesis is reduced. Although cellgrowth may still be observed in this phase, growth is generally verylimited to zero. Gradually cells may loose viability in this phase.

The production of heterologous protein on a medium comprising a commoncarbon source like glucose or another sugar based carbon source issatisfying until limiting conditions start to exist at the end of thefeed phase. Examples of limiting conditions include reduced oxygenconcentration, reduced nutrients like vitamins, carbon, nitrogen, andaccumulation of toxic compounds in the growth medium.

If a fungus, especially a yeast, is grown on a medium comprising glucoseas carbon source, as soon as limiting conditions arise, heterologousprotein production is considerably reduced.

For yeasts grown on common medium including a sugar as carbon source theabove-described phases exist in a fed-batch fermentation. Hence once thedecline phase has started, specific production, which is defined asamount of heterologous protein or peptide produced per gram of biomass,is maintained or reduced. Even although cell density is high, productsynthesis is hence low in the decline phase.

Another disadvantage of common media which often comprise glucose as acarbon source, is the high amount of substrate that is converted tobiomass instead of conversion to product which is usually a heterologousprotein in the context of this invention. Hence in such systems, arelatively high amount of biomass unavoidably accompanies the productionof high levels of heterologous protein.

This high biomass leads to a viscous fermentation medium in which forexample oxygen limitation easily arises.

Therefore there is a desire for a method for heterologous proteinproduction in a host like a fungus which leads to high heterologousprotein yield even under limiting conditions, where normally decline andreduced specific production would exist, whereas at the same time thespecific production of the growth system is maintained or increasedcompared to the known growth systems.

The method of the current invention overcomes at least one of theindicated problems.

SUMMARY OF THE INVENTION

It has surprisingly been found that fungi grown on a medium comprisingethanol as the main carbon source and an inducer like galactose tocontrol the production of a heterologous protein, show high specificproduction of heterologous protein, while specific production of theheterologous protein is surprisingly high even under limitingconditions.

Surprisingly fungi grown according to this method do not show the wellknown characteristics of decline which are encountered for fungi grownon the traditional glucose based media. Under continuation of the feedwith medium comprising this specific carbon source, the fungi maintainor even increase the absolute and specific production level ofheterologous protein.

Therefore the invention relates to a method for the production of aheterologous protein by a fungus comprising growth of said fungus on amedium comprising a carbon source wherein 50-100 wt % of said carbonsource is ethanol, and wherein the medium additionally comprises aninducer.

DETAILED DESCRIPTION OF THE INVENTION

In the context of the invention, the term fungus encompasses yeasts andmoulds.

For the purpose of the invention, the term heterologous protein is meantto include both proteins and peptides.

A heterologous protein is a protein which is not naturally produced bythe fungus but only after the fungus has been modified to this extent.

Where weight percentages are indicated these are weight percentages ontotal product or total medium weight, unless otherwise is indicated.

Where the term oxygen concentration is used, reference is made todissolved oxygen concentration which is measured by the methodillustrated in the examples.

The end of the batch phase is defined as the moment when all carbonsubstrate provided to the cells has been consumed.

Induction phase is defined as the phase which starts after the batchphase from the moment when induction of heterologous protein is starteduntil the moment that maximum induction is obtained for the specificinducer concentration used.

Carbon source is defined as the substrate which provides the supply ofcarbon and energy to the cell. Fungi obtain their cell carbonpredominantly from organic compounds. These commonly serve as bothcarbon source and energy source: they are partially assimilated into thecell material and partially oxidized to provide energy. In this contextreference is made to H. Schlegel in General microbiology, CambridgeUniversity press, 1992, 7^(th) edition, page 194.

The invention relates to a method for the production of a heterologousprotein by a fungus. In order to produce this protein, the fungus isgenetically modified such that controlled production of thisheterologous protein or peptide is possible.

Any suitable construct or transformation method can be used for thisgenetic modification. Examples of suitable constructs and transformationmethods are given in EP-A-481,008 which is herewith incorporated byreference.

In general the modified fungus will after modification comprise a(integrated) vector which comprises the gene encoding the heterologousprotein, under the control of a promoter. The activity of the promoteris regulated by a so called inducer. Examples of promoters include thegalactose promoters like GAL4, GAL7 which are induced by galactose;methanol induced promoters, induced by methanol; ethanol promoters,induced by ethanol; temperature regulated promoters, induced by atemperature change; phosphate regulated promoters, induced by thepresence of phosphate; and glucose repressible promoters, induced by theabsence of glucose.

The fungus is grown on a medium comprising a carbon source of which 50to 100 wt % is ethanol, in combination with the presence of an inducerin the medium.

In the art, the use of ethanol as a carbon source is generallydiscouraged as is exemplified by the disclosure cited below. In summaryuse of ethanol instead of glucose as carbon source is reported to reducethe biomass yield, to require higher oxygen consumption and henceproviding no or limited growth under oxygen limiting conditions.Furthermore ethanol toxicity of strains may lead to loss in viabilityand cell death. This is for example disclosed in table 1 of The yeasts,vol 3 2^(nd) ed chapter 6. Special reference is made to appendix 1 ofthis disclosure which discloses that for yeast, the growth rate onethanol is 0.1 h⁻¹ vs. 0.35 h⁻¹ on glucose. This appendix furthermorediscloses that on a medium with ethanol as carbon substrate, yeasts showa higher oxygen consumption than on a medium comprising glucose ascarbon substrate.

Growth of cells on ethanol as a carbon source is further described byShiba et al (J. of bioscience and bioengineering, vol 89, page 426-430,2000). Shiba disclose expression of carboxypeptidease Y (CPY) usingGAL10 promoter in a Saccharomyces cerevisiae gal80 mutant. The growthmedium comprises ethanol as the sole carbon source. Upon start ofethanol feed, both the growth rate and CPY production are reported todecrease. This system is not under control of an inducer.

The current invention provides a process wherein protein production ismaintained or even increased in the presence of a medium comprising acarbon source of which 50 to 100 wt % is ethanol, especially whenlimiting growth conditions arise. Furthermore the process according tothe invention is applicable for any type of fungus and does not requirespecific gal80 mutants for growth. The process of the invention is aswell controllable via induction.

Saliola, M et al (applied and environmental microbiology, January 1999,p. 53-60) discloses use of the ethanol-inducible KlADH4 promoter forheterologous gene expression in Kluyveromyces lactis. This documentteaches that expression is optimal when ethanol is used as promoter andsimultaneously as sole carbon source in a fed batch system. Thisproduction system does not enable control of gene expression in the feedphase.

A further benefit of the process according to the invention, is controlof heat production which is important for production of heat labileproteins.

It is preferred that the medium comprises a carbon source of which 50 to100 wt % is ethanol, in combination with the presence of an inducer inthe medium, throughout all phases, but is was found that it is possibleto use a medium which does not fulfill these requirements in the batchphase, as long as the requirements are fulfilled by the feed phasemedium.

In a preferred embodiment, the invention relates to a method for theproduction of a heterologous protein by a fungus, which method comprisesa batch phase and a feed phase and wherein the feed phase mediumcomprises a carbon source of which 50 to 100 wt % is ethanol and whereinthe feed phase medium additionally comprise an inducer.

Compared to the known growth systems, which usually contain glucose oranother sugar as the main carbon source, the medium according to theinvention enables high specific production levels even under limitingconditions; i.e. there is preferably no decline phase.

The specific production was even found to be highest in the feed phaseunder limiting oxygen conditions, whereas for glucose based growthsystems, the highest specific production is usually found in the feedphase, where biomass amount is still relatively low.

Without wishing to be bound by any theories, applicants believe that thedecline phase is extended or even absent due to the use of a mediumcomprising a carbon source of which 50 to 100 wt % is ethanol, in themethod of the invention.

In the process of producing a heterologous protein, the feed conditionsare preferably optimised such that cell growth rate and heterologousprotein production are optimal.

As indicated above, if the fungus is grown on industrial scale, a fedbatch system using a fermenter is highly preferred.

In another preferred embodiment, the invention relates to a method forthe production of a heterologous protein by a fungus which methodcomprises a batch phase, an induction phase and a feed phase wherein insaid feed phase

-   -   a) fungal cells are grown to a cell density of at least 5 g/l on        a medium comprising any carbon source, without specific        preference, and subsequently the fungal cells are grown to a        cell density of more than 5 g/l, preferably 10 to 90 g/l, more        preferred 40 to 60 g/l, using a medium comprising an inducer and        a carbon source wherein 50-100 wt % of said carbon source is        ethanol    -   b) after the cell density of step (a) has been obtained,        limiting growth conditions are created,    -   c) after these limiting conditions have set in, the cells are        further grown on a medium comprising a carbon source wherein        50-100 wt % of said carbon source is ethanol.

In this preferred method in a first step cells are grown in a batchphase until carbon substrate has been consumed, and in a second step thecells are grown to a sufficient cell density to enable heterologousprotein production in high amounts. In the first stages of this secondphase, heterologous protein production is induced by addition of aninducer to the feed medium. The time it takes to come from zeroinduction, and thus very low level of heterologous protein production,until induction is maximal, is called the induction phase. Thisinduction phase constitutes the first stage of the feed phase.

Limiting conditions in a fermenter can be obtained in several ways.Limiting conditions are defined as those conditions wherein exponentialcell growth is no longer possible and cell growth is decreasing.

Preferably in the method of the invention, limiting conditions in amedium are created by a method selected from the group comprising

-   -   a) reduction of the oxygen concentration in The fermenter        medium; (e.g. from 0 to below 30%) preferably to below 30%, more        preferred below 15%,    -   b) overfeeding the medium with ethanol until the ethanol level        in the medium is at or above the growth limiting concentration        for the cells of the strain that is grown in the medium    -   c) decreasing the level of other essential ingredients for        growth of the cells, said ingredient preferably being selected        from nitrogen, phosphor, sulphur and vitamins.

Preferably after the batch phase the feed profile is exponentiallyincreasing with biomass production. When oxygen limitation arises andthe decline phase is entered, the feed rate is preferably set to a rateas to maintain an ethanol concentration of below 10 vol % in thefermenter medium. This can for example be obtained by a linear feed rateor a pulsed feed rate or a step wise feed rate.

In an even more preferred embodiment, the method of the invention iscarried out as a repeated fed batch process.

Preferably the inducer is suitable for turning on the promoter which isoperably connected to the heterologous gene in the gene construct usedfor transformation of the host fungus. Preferred inducers are galactose,methanol, temperature, and phosphates.

The most preferred inducer is galactose.

The current invention does not relate to expression methods whereinethanol is both used as an inducer and as (part of) the carbon source.

When galactose is the inducer, the amount of galactose in the mediumshould preferably be such that the promoter is turned on to the desiredlevel while the amount is so low, that galactose is not metabolised. Toprevent this, it is possible to use a strain which is unable tometabolise the inducer.

Preferably the composition of the feed medium is such that the medium inthe fermenter comprises from 0.1 to 10 wt % galactose, more preferredfrom 0.05 to 1 wt %, even more preferred from 0.05 to 0.2 wt %galactose.

The carbon source in the medium according to the invention comprises atleast 50 wt % and up to 100 wt % ethanol. Preferably the carbon sourcecomprises from 80 to 100 wt % ethanol. The remainder of the carbonsource can for example be a sugar such as glucose, galactose, lactose,sucrose, fructose or another compound like glycerol, acetate, or complexcarbon substrates like whey and molasses.

The fungus can be a yeast or a mould. Examples of suitable yeast generainclude Saccharomyces, Kluyveromyces, Pichia, Hansenula. Examples ofsuitable moulds include Aspergillus, Rhizopus, Trichoderma.

The especially preferred organism is Saccharomyces cerevisiae.

The heterologous protein can be any protein or peptide of whichproduction is desired. The method according to the invention was foundto be especially suitable for production of antifreeze peptides,antibodies or fragments thereof, or enzymes such as cutinase andgalactosidase.

Antifreeze peptides are proteins that have the ability to modify thegrowth of ice, and are for example described in biotechnology advancesvol. 13, no 3, pp 375-402, 1995 by Griffith et al, which is herewithincorporated by reference.

The medium for the various phases in the growth of the fungus generallycomprise a carbon source, optionally an inducer, who's presence isrequired after the batch phase, and optionally further ingredients suchas vitamins, yeast extract, trace metal ions, acidification agents tokeep the pH at a desired level, phosphate salts, sulphate salts water,and antifoaming agent.

According to another preferred embodiment the invention relates to amethod for producing a heterologous protein or peptide wherein the batchmedium comprises from 1-40 wt % glucose, water, trace metals, optionallyan antifoaming agent, yeast extract, vitamins, phosphate salts andsulphate salts and wherein the feed medium comprises from 5 to 35 vol %ethanol, 0.1-10 wt % galactose, water, trace metals, and optionallyantifoaming agent, yeast extract, vitamins, phosphate salts and sulphatesalts and wherein the “feed rate” “φ” is from 0.25 to 4 g/min on 10litre scale or a corresponding value for a larger scale fermentation.

The invention furthermore relates to a heterologous protein isolate,especially antifreeze peptide isolate, obtained by the method accordingto the invention.

The invention will now be illustrated by the following non-limitingexamples.

EXAMPLES Media and Cultivation

All media and cultivation data is based on 10 litre scale ethanolfermentation.

For 10 m³ scale it's roughly the same adapted to scale factor 1000.

Media Preculture Media:

INOCULUM MEDIA Compound g/l Supplier YNB YNB w/o amino acids 6.7 DifcoYeast Glucose*1aq 5 AVEBE nitrogen In demineralised water base YPD YE 10Difco Yeast bacto pepton 20 Difco peptone Glucose*1aq 20 AVEBE dextroseIn demineralised water

Batch and feed media are listed in table 1 for 10 litre scale

TABLE 1 Concentration (g/kg) Feed Comp. Feed Example Compound SupplierBatch Ethanol Glucose Glucose Avebe 22.0 — 440.0 Tap water EtOH* Lamers& — 333.84 — Pleuger Tap water Galactose HMS — 3.0 3.0 Tap water YE KatG Ohly 10.0 25.0 25.0 KH₂PO₄ Acros 2.1 12.0 12.0 MgSO₄•7H₂O Merck 0.62.5 2.5 Egli vitamins See Table 2 1.0 2.0 2.0 Egli trace See Table 210.0 20.0 20.0 metals Structol J Schill & 0.4 0.8 0.8 673 Seilacher Tapwater Total weight 5,500 4,000 4,000 (g) *Ethanol pure, 96.2% v/vcontent, non denatured All media were sterilised 25 minutes at 121°C./1.2 bar (Linden autoclave), sugars separately. The tap water amountsare described below and total amount is such that total weight isindicated in the bottom row of table 1.described below and total amount is such that total weight is asindicated in the bottom row of table 1.

Egli Vitamins and Trace Metals Composition

TABLE 2 Egli vitamins (1000 × Egli trace metals (100 × stock solution)stock solution) Compound g/l Compound g/l Thiamine (HCl) 5.00 CaCl₂•2H₂O5.50 Meso-inosit 47.00 FeSO₄•7H₂O 3.73 Pyridoxine 1.20 MnSO₄•1H₂O 1.40Panthotenic 23.00 ZnSO₄•7H₂O 1.35 Biotine 0.05 CuSO₄•5H₂O 0.40CoCl₂•6H₂O 0.45 Vitamins were 0.22 μm filter sterilized

Strain

The strain used was Saccharomyces cerevisiae CEN.PK102-3A. The basicCEN.PK2 S. cerevisiae strain is commercially available from EUROSCARF,Institute for Microbiology, Johann Wolfgang Goethe-University Frankfurt,Marie-Curie-Strasse 9; Building N250, D-60439 Frankfurt, Germany.

This S. cerevisiae strain is not able to metabolise galactose as theGAL1 gene has been disrupted by insertion of sequences derived form theS. cerevisiae URA3 gene. The host strain was transformed with anexpression plasmid comprising the following elements:

-   -   Promoter: GAL7 promoter, leader GAPDH. GAL7 promoter: two        upstream activating sequences are always present. They are 2        natural elements that are part of the sequence as attached.    -   Selection marker: Leu2d    -   Signal sequence: invertase (SUC2)    -   Integration target: fragment of rDNA repeat with unique        restriction site for targeting integration to a specific region        in yeast chromosome XII.    -   Heterologous gene:

Example 1

An Antifreeze Protein Gene Encoding the Ocean Pout HPLC12 AntifreezeProtein (WO-A-9702343)

Example 2

K609B, an Antibody Against Virulence Factors in Piglets

Example 3

Protein VHH G which is HGL11, which is a Heavy Chain Immunoglobulin; theLipase Inhibitor Against Human Gastric Lipase Disclosed inEP-A-1,134,231

Example 4

Protein VHH P which is HPL18, which is a Heavy Chain Immunoglobulin; theLipase Inhibitor Against Human Pancreas Lipase Disclosed inEP-A-1,134,231.

The proteins of example 2-4 are antibodies obtained by immunisation of allama according to the procedures disclosed in EP-A-1,134,231.

Cultivation Strain Storage

Strains were stored at −80° C. in single batch from YNB-grown culturesdiluted 1:1 with a mixture of skimmed milk and 20% glycerol.

Inoculation

50 ml YNB was inoculated with 1 ml stored strain, and incubated for 48hours ±2 hours at 30° C. at 150 r.p.m.

Subsequently the inoculum was transferred to 500 ml of 2% YPD, followedby incubation for 24 hours ±2 hours at 30° C. at 150 r.p.m.

Fermenter

The fermentations were performed in standard fermenters with a workingvolume of ten litres. For temperature control, the fermenter wasequipped with a cooling coil and a heating finger. Baffles were ofstandard dimensions. A Rushton type impeller with six blades was usedfor stirring. Dissolved oxygen (DO₂) was measured with an Ingold®DO₂-electrode (Mettler-Toledo) and the pH was measured with an Ingold®Impro 3000 gel electrode (Mettler-Toledo). A mass spectrophotometerPrima 600 (VG gas analysis systems) was used for measuring the offgas.The whole fermentation process was automated and software-controlled butcould as well be carried out manually on the basis of the guidanceprovided below.

A feed profile was imposed to control the fermentation. pH wascontrolled using 3M phosphoric acid (Baker) and 12.5% v/v ammonia(Merck).

DO₂ was controlled at 30% by automatic adjustment of the impeller speeduntil maximum stirrer speed occurred (1000 rpm).

During fermentation 5 ml samples were taken and cooled at 4° C. with anautosampler for dry weight determination and product concentrationanalysis.

Batch Phase

To start the batch phase 500 mL of YPD inoculum was added to 5.5 L batchmedium. Fermentation parameters were as according to table 3.1 When theethanol content in the offgas decreases to 300 ppm the exponential feedwas started.

Feed Phase

The feed medium was separated into two feed bottles connected to thesame pump.

One feed bottle contained the ethanol and tap water to 2 Litres and wasfed to the fermenter through the bottom plate.

Second feed bottle contained all other feed components and water to 2Litres and was fed through the top plate.

From both bottles, connected to one pump, the same exponential feed ratewas applied according to equation 1. For two bottles the resulting feedrate is twice the pumprate.

$\begin{matrix}{\Phi_{v,t} = {\frac{\mu*X_{0}*{\mathbb{e}}^{\mu*t}}{\rho_{feed}*Y_{X,S}*60}\mspace{14mu}\left\lbrack {g\text{/}\min} \right\rbrack}} & \left( {{equation}\mspace{14mu} 1} \right)\end{matrix}$

Φ_(v,t) = feed rate g/min μ = growth rate h⁻¹ X₀ = biomass at feedstartg (Mw = 24.6 g/mol) t = time since feedstart min ρ_(feed) = feed densityg ethanol/g feed Y_(x,s) = estim. yield of biomass g X/g substrate 60 =time factor min/h

Feed parameters were according to table 3.

TABLE 3 Fermentation feed parameters for 10 liters ethanol fermentationParameter Value μ (l/h) 0.06 X₀ (mol) 1.06 ρ_(feed) (g/g) 0.41 Y_(xs)(g/g) 0.45 T batch (° C.) 30 T feed (° C.) 21 Airflow batch (l/min) 2Airflow feed (l/min) 6 DO₂ (%) 30 DO₂ minimum (%) 0 PH 5 SS min (rpm)300 SS max (rpm) 1000 EtOH criterium 300 feedstart (ppm) EtOH criterium800 declinestart (ppm) EtOH maximum (ppm) 2000 Initial total feed 0.28g/min rate (g/min) Linear total feed   2 g/min rate (g/min)

When the oxygen concentration was 0%, the limiting conditions were setin and the pumprate was set to linear feed rate. This feeding wascontinued until all feed was depleted.

Results Example 1

Ethanol as carbon source AFP (mg/kg Dry Specific Time fermentationmatter production* (hr) broth) (g/kg) (mg/g) 0 0 2.4 0 3 0 3.4 0 6 0 5.70 9 0 7.6 0 12 0 10.9 0 15 0 10.5 0 18 0 11.7 0 21 0 13.3 0 24 0 15.6 027 7.0 17.9 0.393 30 11.4 20.7 0.549 33 17.9 24.8 0.721 36 24.2 28.80.840 39 32.4 32.7 0.990 42 41.8 38.4 1.089 45 52.4 42.7 1.227 48 66.949.0 1.366 51 78.7 55.0 1.432 54 93.7 60.6 1.546 57 112.2 64.8 1.732 60132.1 68.6 1.925 63 151.2 71.9 2.104 66 165.8 75.5 2.195 *Specificproduction: mg AFP/g dry matter

On the basis of these results it is concluded that production of AFPwhen in the feed phase after limiting conditions, arise, the feedingwith medium containing a carbon source which is 100 wt % ethanol iscontinued, leads to surprisingly high specific production of AFP.

It was observed that the biomass production was constant without loss ofabsolute AFP productivity.

Comparative Example

Growth medium in feed phase did not contain ethanol but 100 wt % glucoseas carbon source. The medium for this experiment is described above.Also the fermenter feed conditions are specified above with thefollowing exceptions:

-   -   The feed medium was applied to the fermenter from one feed        bottle, containing all components and was fed to the fermenter        through the bottom plate. An exponential feed rate was applied        according to eq 1.    -   Feed parameters were according to table 3 with exception for the        X0-value which was set to 2.11 mol to result in the same feed        rate with one feed bottle as in the ethanol fermentation with        two feed bottles on one pump.

The results were as follows:

Dry Specific Time AFP matter production* (hr) (mg/kg) (g/kg) (mg/g) 0 01.9 0 3 0 3.3 0 6 0 5.5 0 9 0 7.2 0 12 0 10.4 0 15 0 11.4 0 18 0 13.3 021 0.8 15.8 0.049 24 2.6 18.4 0.142 27 5.8 20.3 0.286 30 10.3 24.6 0.42133 16.8 28.6 0.588 36 25.5 33.0 0.773 39 35.4 37.5 0.942 42 46.8 43.41.077 45 60.3 50.0 1.204 48 74.4 55.9 1.331 51 90.8 62.9 1.445 57 126.479.2 1.596 60 135.2 79.5 1.701 63 135.13 79.3 1.705

It is concluded that when the growth medium contains only glucose as thecarbon substrate, AFP production is decreased up from the moment whenlimiting conditions set in. AFP production no longer increases and theproduction declines.

Results Example 2

Glucose as carbon source Ethanol as carbon source (comparative example2) VHH VHH Specific (mg/kg Dry Specific (mg/kg Dry produc- fermentationmatter production* fermentation matter tion* broth) (g/kg) (mg/g) broth)(g/kg) (mg/g) 0 0.91 0 0 6.63 0.00 0 6.17 0 0 12.34 0.00 0 11.66 0106.33 25.60 4.15 141.77 21.26 6.67 248.09 35.65 6.96 927.40 48.68 19.05425.30 45.02 9.45 1063.26 51.65 20.58 673.40 57.59 11.69 1222.75 56.6821.57 856.52 72.22 11.86 1151.87 55.31 20.83 915.59 71.31 12.84*Specific production: mg heterologous protein/g dry matter

It is concluded that the specific production of heterologous proteinusing ethanol as carbon source is much higher than on glucose as carbonsource.

Results Example 3

Glucose as carbon source Ethanol as carbon source (comparative example3) VHH VHH (mg/kg Specific (mg/kg Specific fermen- Dry produc- fermenta-Dry produc- Time tation matter tion* Time tion matter tion* (h) broth)(g/kg) (mg/g) (h) broth) (g/kg) (mg/g) 3 0 2.87 0 21 103.45 17.19 6.02 60 5.73 0 27 127.59 20.78 6.14 9 0 6.93 0 33 227.59 28.66 7.94 33 268.9745.37 5.93 39 272.41 36.78 7.41 36 417.24 50.86 8.20 45 327.59 47.526.89 39 537.93 54.45 9.88 51 413.79 57.07 7.25 42 689.66 56.36 12.24 57503.45 64.48 7.81 63 565.52 67.10 8.43 69 596.55 69.73 8.56 *Specificproduction: mg heterologous protein/g dry matter

It is concluded that the specific production of heterologous proteinusing ethanol as carbon source is much higher than on glucose as carbonsource.

Results Example 4

Glucose as carbon source Ethanol as carbon source (comparative example4) VHH VHH (mg/kg Specific (mg/kg Specific fermen- Dry produc- fermenta-Dry produc- Time tation matter tion* Time tion matter tion* (h) broth)(g/kg) (mg/g) (h) broth) (g/kg) (mg/g) 0 0.00 0.72 0.00 15 13.79 10.511.31 12 0.00 9.79 0.00 21 27.59 16.00 1.72 18 13.79 17.19 0.80 27 55.1719.10 2.89 24 155.17 25.31 6.13 33 117.24 25.07 4.68 30 275.86 35.827.70 39 155.17 32.00 4.85 36 403.45 46.80 8.62 45 213.79 45.13 4.74 39472.41 50.63 9.33 51 210.34 56.83 3.70 42 620.69 53.49 11.60 57 255.1768.30 3.74 45 675.86 58.03 11.65 *Specific production: mg protein/g drymatter

It is concluded that the specific production of heterologous proteinusing ethanol as carbon source is much higher than on glucose as carbonsource.

1. A method for the production of a heterologous protein by a funguswhich method comprises a batch phase, an induction phase and a feedphase, said batch phase comprising growing fungal cells to a celldensity of at least 5/gl on a batch medium comprising any carbon source,without specific preference; and said induction phase and feed phasecomprising: (a) subsequently growing the fungal cells to a cell densityof from 10 to 90 g/l, using a feed medium comprising an inducer and acarbon source wherein 50-100 wt. % of said carbon source is ethanol;then (b) creating limiting growth conditions by a method selected fromthe group comprising reducing the oxygen concentration in the fermentermedium to from 0 to below 30%; overfeeding the medium with ethanol untilthe ethanol level in the medium is at or above the growth limitingconcentration for the cells of the strain that is grown in the medium;or decreasing the level of other essential ingredients for growth of thecells said ingredients being selected from nitrogen, phosphor, sulfurand vitamins; and then (c) further growing the cells on a mediumcomprising a carbon source wherein 50-100 wt. % of said carbon source isethanol; with the proviso that the method does not relate to expressionmethods wherein ethanol is both used as inducer and as the carbonsource.
 2. The method according to claim 1 wherein the method is carriedout in a fermenter, which is fed with feed medium at a feed rate whichis such that the ethanol concentration in the fermenter is maintainedbelow 10 vol %.
 3. The method according to claim 1 further comprisescarrying out the method as a repeated fed batch process.
 4. The methodaccording to claim 1 wherein the inducer is selected from the groupcomprising galactose, methanol, temperature and phosphates.
 5. Themethod according to claim 1, wherein the feed medium comprises from 0.1to 10 wt % galactose.
 6. The method according to claim 1, wherein thecarbon source comprises 80-100 wt % ethanol.
 7. The method according toclaim 1, wherein the fungus is a yeast.
 8. The method according to claim1 wherein the heterologous protein or peptide is selected fromantifreeze peptides, antibodies or fragments thereof, and enzymes. 9.The method according to claim 1 wherein the batch medium comprises from1-40 wt. % glucose, water, trace metals, and wherein the feed mediumcomprises from 5 to 35 vol. % ethanol, 0.1-10 wt. % galactose, water,trace metals, and optionally antifoaming agent yeast extract, vitamins,phosphate salts and sulphate salts and wherein the “feed rate”“φ” isfrom 0.25 to 4 g/min on 10 liter scale.
 10. The method according toclaim 1 wherein the batch medium further comprises an antifoaming agent,yeast extract, vitamins, phosphate salts and sulphate salts.
 11. Themethod according to claim 7 wherein the yeast is Saccharomycescerevisiae.
 12. The method according to claim 1 wherein in step (a) thefungal cells are grown to a cell density of from 40 to 90 g/l.